Pcmv6 anxa10 gfp

Pre-designed and validated Cul4A (Dharmacon, Lafayette, CO, USA), ANXA10 (Santa Cruz, Santa Cruz, CA, USA), and universal negative control siRNAs were transfected (final concentration = 50 nM) in cells grown to 80% confluence on 6-well plates using an antibiotic-free media and Lipofectamine™ RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. At 96 h after transfection, the cells were treated with gemcitabine for 72 h followed by counting for viable cells. The pCMV6-ANXA10-GFP (OriGene, Rockville, MD, USA) and empty pCDNA3 (Invitrogen, Carlsbad, CA, USA) vectors were transfected with OmniFect™ transfection reagent (TransOMIC, Huntsville, AL, USA), following the manufacturer’s instructions. Cells were plated in 6-well plates in antibiotic-free media and then transfection was performed with cells at 80% confluence, with a final concentration of 0.5 μg for each vector.

Then, 293T cells were transiently co-transfected with pBabe-Cul4A-myc-his and pCMV6-ANXA10-GFP (OriGene, Rockville, MD, USA) vectors using Lipofectamine 2000 transfection reagent (Invitrogen). Twenty-four hours after transfection, the cells were treated with 10 μg/mL of MG132 (Sigma) for 24 h, and then harvested in a NP-40 lysis buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 1% NP40), protease inhibitor, and phosphatase inhibitor cocktail (Roche, Lewes, UK, USA). Immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer’s protocols. Anti-GFP (OriGene) and Anti-Cul4A (Abcam) antibodies were used for immunoprecipitation.

The 293T cells were co-transfected with a combination of pBabe-Cul4A-myc-his and pCMV6-ANXA10-GFP (OriGene) with or without pRK5-HA-Ubiquitin-WT (Addgene, Cambridge, MA, USA). All the cells were treated with 10 µg/mL of MG132 for 24 h prior to lysis. Anti-GFP antibody was used for immunoprecipitation. Anti-HA tag antibody (Cell Signaling, Danvers, MA, USA) was used for the western blot analysis.